The overarching aim in this proposal is to determine at atomic resolution the structural basis of glucose transporter (GLUT) function. This will reveal, at a molecular level, its transport mechanism and involvement in pathologies of diabetes, cancer and other diseases. It will also provide a rational platform for developing both novel drugs and medical detection methods. GLUT carries carbohydrates (glucose, fructose, galactose and others), major energy sources for living cells, across membranes. So far fourteen human GLUTs have been identified and characterized. They exhibit significant variability in function and tissue expression despite their relatively high sequence similarity. Determining the three-dimensional (3D) structure of any GLUT will be a remarkable breakthrough in the study of this fundamental transport system that wil open new research avenues. Structural information in conjunction with site-directed mutagenesis and functional studies will allow us to pinpoint the molecular base of the functional variability of GLUT members, so that designed ligands for a particular GLUT can be rationally pursued. To understand the functional diversity of glucose transporters and increase the chances of obtaining a structural solution, the targets include: human glucose transporters (hGLUTs), and several bacterial counterparts with greater than 25% identity and 45% homology to hGLUTs amino-acid sequences. Milligram quantities of purified, functional protein for seven bacterial and three human GLUTs were produced. Thus far, crystallization efforts have been successful with four bacterial GLUTs among which two produced diffracting crystals; crystals of one bacterial GLUT diffracted up to 3.1 Angstroms resolution. Our hypotheses are that 1) the 3D structure of bacterial GLUTs will be representative for those of human GLUTs, as the active site residues are conserved from prokaryotic to eukaryotic species, and 2) as a MFS member, GLUT has two symmetric 6-helicies bundles and a hydrophilic internal cavity which undergo conformational changes during the transport mechanism. Our two specific aims are to: 1a) Determine the 3D structures of those bacterial GLUTs that yield well-diffracting crystals. 1b) Produce diffracting crystals of at least one purified human GLUT. 2a) Investigate the transport of purified GLUTs. 2b) Develop high-throughput binding assay for GLUTs in order to find novel inhibitors. The results of our proposed studies will impact our fundamental understanding of GLUTs. Furthermore, they will empower the search for novel drugs for the treatment of diseases involving GLUTs in two ways: 1) structure determination of GLUTs will guide rational drug design; 2) identification of novel ligands through high- throughput screening of small molecules will provide lead compounds for new drugs.